PROJECT SUMMARY/ABSTRACT The objective of this proposal is to develop an efficient and robust method to epigenetically reprogram nave human T cells into regulatory T cells (Tregs) that can prevent graft versus host disease (GvHD). Our short term goal for Phase I of the STTR award is to demonstrate that we can epigenetically-reprogram nave T cells using CRISPR/Cas9-based technology into immunosuppressive cells that have the protein marker, gene expression, and epigenetic regulatory profiles of Tregs. This will motivate Phase II of the STTR, where we will test the epigenetically-reprogrammed Tregs in a humanized mouse model of graft versus host disease. The data obtained during the STTR will position the therapy for clinical trials to prevent GvHD in hematopoietic cell transplantation. There is a clinical need to improve patient outcomes in allogeneic hematopoietic cell transplantation (HCT) due to the high incidence, morbiditity, and mortality associated with GvHD. Approximately half of all HCTs utilize hematopoietic cells from unrelated donors, and approximately 26,800 patients will undergo this procedure per year worldwide. Despite improved diagnostics to match the most immunogenic antigens on the donor cells with those of the recipient, up to 70% of allogeneic HCT recipients will develop GvHD as a result of their graft. Only 50% of HCT recipients that develop GvHD survive 3 years beyond the transplant procedure. GvHD is mediated by donor T cells that recognize polymorphisms in the protein-coding genome of the graft and the recipient, and these reactive T cells can be suppressed by Tregs. We will utilize CRISPR/Cas9-based epigenetic editing tools to reprogram nave human T cells, which be easily collected from whole blood by apheresis or density gradient centrifugation, into immunosuppressive Tregs. We will first perform a high-throughput screens of candidate Treg- specific gene regulatory elements that we have identified through computational analysis of epigenomic data. We will then validate that these elements can be activated by CRISPR/Cas9-based epigenome modification tools that we have developed to generate T cells with the phenotype, in vitro suppressive properties, gene expression, and chromatin regulatory profile of Tregs. !